Cerebral deposition of the amyloid-beta protein (A-beta) in the form of fibrils is intimately involved in the pathogenesis of Alzheimer's disease (AD). An unidentified protease called gamma-secretase catalyzes the final step in the generation of A-beta cleaving within the single transmembrane region of the amyloid precursor protein (APP). Numerous mutations in presenilins 1 and 2 (PS1 and PS2) cause AD, and these mutations alter gamma-secretase activity to increase levels of a highly fibrillogenic 42-residue variant of A-beta (A-beta42) over the more abundant 40-residue variant A-beta40. Cells deficient in PS1 have dramatically reduced gamma-secretase activity, indicating that PS1 mediates the major portion of this proteolytic event. Through inhibitor studies and molecular modeling, the principal investigator has found that gamma-secretase has properties of an aspartyl protease and likely catalyzes an unusual intramembranous proteolysis. Provocatively, two conserved transmembrane aspartates in PS1 are each critical for both gamma-secretase cleavage of APP and the normal endoproteolysis of PS1. These and other findings are consistent with the hypothesis that presenilins themselves are gamma-secretases, intramembrane-cleaving aspartyl proteases activated through autoproteolysis. The objective of the present proposal is to understand the biochemistry of gamma-secretases, how presenilins mediate this proteolytic activity, and how AD-causing mutations in APP and presenilins affect this proteolysis to specifically increase A-beta42. Questions to be addressed include: (1) Do gamma-secretase inhibitors bind to presenilins? Does gamma-secretase and/or presenilins recognize a helical conformation of the APP TM region? Can purified presenilins catalyze hydrolysis? (2) The Notch receptor, involved in cell fate decisions during development, undergoes proteolytic processing strikingly similar to that of APP, including an apparently intramembranous cleavage event. Is this final proteolysis catalyzed by a gamma-secretase-like enzyme? Does PS1 mediate this proteolysis? (3) Are separate gamma-secretases responsible for A-beta40 and A-beta42 formation? How do FAD-mutant APP and presenilins alter gamma-secretase processing to increase A-beta42 production?